How to inoculate culture media

InoculationInoculation From Latin word “Inoculare” which means to implant

or to introduce.

It means to implant or introduce microorganisms or

infectious material into a culture medium for growth

of microorganisms.

Aseptic techniques should always be followed while

inoculating;

1. Media in petri dishes.

2. Slope media (Agar Slants).

3. Inoculation of stab media (deeps).

4. Inoculation of fluid media.

Aseptic TechniquesAseptic Techniques Aseptic technique refers to a procedure that is

performed under sterile conditions to prevent any

contamination e.g. use of flame.

Aseptic procedures can be done in a number of

ways:

1. Use of Bunsen burner or spirit lamp to sterilize wire

loops/inoculating needles before and after use (when

loop turns red hot it become sterilized).

2. Flame the necks of specimen bottles, culture tubes

after removing and before replacing caps.

3. While inoculation, do not let the caps of tubes to touch

an unsterile surface. This can be avoided by holding the cap

in the hand.

4. Decontaminate the work place before starting the

day’s work and after finishing (by using 70% ethanol or spirit

etc).

5. Use of a safety cabinet when working with hazardous

pathogens.

6. Wear protective clothing, wash the hands after

handling infected material and never mouth pipette any

solvent, eat, drink in the laboratory.

Aseptic Removal of Microorganisms from a Broth Culture

Transferring Microorganisms into a Broth Tube

Aseptic Removal of Microorganisms from a Plate Culture

11.. Inoculation of media in petri Inoculation of media in petri dishesdishes

Plating out or looping out is the term used

for inoculating media

in a petri dish.

Using a sterile loop, apply the inoculum to a

small area of the plate (the ‘well’).

Flame sterilize the loop. When cool, spread

the inoculum. Each time flame sterilize

the loop.

The technique used to inoculate media in

petri dishes (plates) must:

a.Provide single colonies for identification.

b.It must also show whether a culture is pure

or mixed, i.e. consisting of a single type of

organism or several different organisms.

A pathogen must be isolated in pure

culture before it can be identified and

tested for antimicrobial sensitivity.

Inoculation of a plate of culture medium to give single colonies

Simplified technique of inoculating a plate of culture medium

Inoculation of half a plate of culture medium

Different ways of inoculating a third of a plate of culture medium

22. Inoculation of SlopesInoculation of Slopes

To inoculate slopes such as Dorset egg medium or

Loeffler serum, use a sterile straight wire streak the

inoculum down the centre of the slope and then

pull the wire and spread the inoculum in a zigzag

pattern on the slope surface.

To inoculate a slope (slant) and butt medium, such

as triple sugar iron agar, use a sterile straight wire

to stab into the butt first and then use the same

wire to streak the slope in a zig-zag pattern.

3. 3. Inoculation of Stab media (deep Inoculation of Stab media (deep

tube)tube)

Use a sterile straight wire to inoculate a stab

medium.

Stab through the centre of the medium taking

care to withdraw the wire along the line of

inoculum without making further stab lines.

44. Inoculation of fluid mediaInoculation of fluid media

Using a sterile wire loop, obtain a sample of microbial

culture (a small amount of bacterial colony).

Hold the bottle or tube to be inoculated at an angle

and rub the loop against the side of the container

below the level of the fluid.

Growth can be observed in the inoculated tube as

turbidity or milky appearance after incubation at 370C

for 24 hours.

Inoculated Broth (Turbid) Un-inoculated Broth (No Growth)